Background Osteosarcoma is the most common pediatric bone cancer in the United States. Due to advances in surgery and chemotherapy, cure rates for patients with localized disease approach 70%. However, long-term survival for patients with metastatic disease is only 25% and multiple attempts at intensification of chemotherapy have not improved this dismal survival rate. These figures point to the need to develop new therapeutic regimens. A different treatment approach is to utilize inhibitors that target activated tyrosine kinases. Imatinib, which targets ABL and KIT, has been the paradigm for such treatment. Patients with CML have activation of ABL due to a BCR-ABL translocation and patients with GIST have activating mutations in KIT. Both groups of patients have had remarkable responses when treated with Imatinib. A similar approach to treat patients with osteosarcoma does not exist because there has not been a systematic examination of mutations in tyrosine kinases. Our goal is sequence every gene in osteosarcoma tumor samples to try to identify activating mutations. In 2009, in preparation for this project, we identified tumor specimens collected from patients previously treated at the NIH under Pediatric Oncology Branch protocols. Approval was granted from the Office of Human Subjects Research to perform sequencing analysis using these samples. We isolated DNA and RNA and went on to determine the quality of these samples. At the same time a small portion of the tumor was sent for sectioning and H+E slides were examined with a pediatric pathologist, verifying the presence of tumor and the diagnosis of osteosarcoma. Results from 2010 In 2010, in preparation of evaluation of patient samples for sequencing, we utilized a panel of well-established osteosarcoma cell lines and evaluated them by comparative genomic hybridization. In addition, we also analyzed cell lines that were recently derived at the NIH from patients at the time of their definitive resection. The results suggested that all of our samples were consistent with osteosarcoma and that they were of sufficient purity to undergo next generation sequencing. We have also published two review articles detailing the biology of osteosarcoma and our plans to further understand its pathogenesis. Strategies to explore new approaches in the investigation and treatment of osteosarcoma published in Cancer Treatment and Research and Stratifying osteosarcoma: minimizing and maximizing therapy published in Current Oncology Reports. Plans for 2011 In 2011, in collaboration with members of Dr. Paul Meltzers laboratory, we will perform next generation sequencing of osteosarcoma cell lines. The results of this analysis will better prepare us for sequencing of DNA obtained from patient tumor samples. Significance and Goals The successful completion of the above objectives may reveal the mechanism, or mechanisms, that underlie the initiation and progression of osteosarcoma. The finding of genetic changes will then allow us to perform molecular and biological evaluations to determine if they are relevant targets that merit further evaluation. Papers and Presentations in 2010 Kim SY, Helman LJ. Strategies to explore new approaches in the investigation and treatment of osteosarcoma. Cancer Treat Res 2010;152:517-528. PMID 20213413. Niswander LM, Kim SY. Stratifying osteosarcoma: minimizing and maximizing therapy. Curr Oncol Rep 2010;12:266-270. PMID 20473649.